产志贺毒素样大肠杆菌F18ab菌毛操纵子基因的体外非诱导系统的表达和鉴定

Cloning and expression of F18 fimbrial operon gene cluster from Shiga-like toxigenic E.coli(SLTEC) using non-induced expression system

以产志贺毒素样大肠杆菌(SLTEC)F18ab血清型标准菌株107/86基因组DNA为模板,利用PCR技术成功扩增出编码F18ab完整菌毛操纵子fed基因,克隆入表达载体pBR322,经限制性内切酶酶切分析,DNA琼脂糖电泳鉴定并结合序列测定分析,构建和筛选出含fed完整基因正确插入的pBR322-fed重组质粒,将上述重组质粒转化至不含任何菌毛结构的大肠杆菌SE5000,该表达重组菌能分别与兔抗F18ab亚单位蛋白FedF高免血清、鼠抗F18ab菌毛a单因子单克隆抗体、兔抗F18ab菌毛高免血清和抗F18ab菌毛IgG抗体产生明显的凝集反应。用热抽提法分别抽提和纯化SLTEC F18ab标准株107/86和重组菌SE5000(pBR322-fed)体外表达的F18ab菌毛,纯化菌毛经SDS-PAGE电泳和考马斯亮蓝染色获单一相对分子质量约为15 000蛋白条带。Western-blotting结果表明:兔抗F18ab菌毛高免血清能特异性识别SLTEC F18ab标准株107/86和重组菌SE5000(pBR322-fed)所提纯的单一主要结构蛋白。用重组菌SE5000(pBR322-fed)进行易感仔猪小肠上皮细胞体外黏附试验和黏附抑制试验,结果表明:重组菌SE5000(pBR322-fed)和SLTEC F18ab标准株107/86一样具有较强的黏附易感仔猪小肠上皮细胞的能力,而兔抗F18ab菌毛高免血清能有效地抑制上述重组菌SE5000(pBR322-fed)和SLTEC F18ab标准株107/86对易感仔猪小肠上皮细胞的黏附结合。

The fed operon gene cluster encoding the F18ab-serotype fimbriae was amplified by Expand Long Template PCR using the genomic DNA template from Shiga-like toxigenic E.coli reference strain 107/86.The predicted PCR products with the restriction enzyme sites at each end were digested and then cloned into the expression vector pBR322,the recombinant plasmid pBR322-fed with the inserts of the whole fed gene clusters were constructed and screened,further confirmed by the means of combination with both restriction endonuclease analysis and sequencing.The fimbriae F18ab were isolated and purified from the recombinant E.coli SE5000（pBR322-fed）,and only a single major band of protein with size of approximately 15 000 was visualized in Coomassie blue-stained gels after SDS-PAGE,and this protein band was recognized positively by the polyclonal sera against fimbriae F18ab in Western-blotting assay.The results in agglutination assay showed that the polyclonal sera,purified IgG,and monoclonal antibody（Mab） directed against the major subunit of F18ab fimbriae（FedF）,F18ab fimbriae from the reference strain E.coli 107/86 reacted to the recombinant E.coli SE5000 expressing the fimbriae F18ab,respectively.Small intestine epithelial cells from the susceptible piglets with the genotypes of FUT1 gene M307GG were prepared and tested for the adherence to the recombinant E.coli SE5000（pBR322-fed） under the microscopic examination.Adhesion and adhesion inhibition test showed the recombinant E.coli SE5000（pBR322-fed） could adhere to the jejunal epithelial cells in vitro as the reference strain E.coli 107/86 did.The polyclonal anti-sera directed against fimbriae F18ab can efficiently inhibit the fimbriae-mediated susceptible piglet jejunal epithelial cells adherence to the recombinant E.coli SE5000（pBR322-fed） and reference strain E.coli 107/86.